We recently reported that the majority of cases of Alexander disease are associated with missense mutations in the coding region of the GFAP gene, which encodes an astrocyte specific intermediate filament. The putative disease-causing mutations are all heterozygous, and likely act in a dominant gain-of-function fashion. To prove that these mutations are causal for the disease, assess potential genotype-phenotype correlations, and provide an in vivo model for studies of pathogenesis, we propose generating mice carrying the same mutations as those identified in human patients. In Specific Aim 1 we will engineer targeting vectors that will be used to introduce point mutations into the coding of GFAP, based on four different mutations found in Alexander disease patients (R79H, R239H, R239C, and R416W). In Specific Aim 2 we will generate targeted clones of embryonic stem cells that are heterozygous for GFAP point mutants, excise the neo selectable marker by Cre/lox technology, and produce mice carrying these mutations by blastocyst injections. In Specific Aim 3 we will evaluate the phenotype of the mutant mice using molecular, pathological, physiological, and behavioral assays. These studies promise novel information on the pathological significance of mutant intermediate filament expression in astrocytes, and will suggest mechanisms by which primary astrocyte dysfunction leads to generalized CNS disease.